Hepoxilins and modulators of ichthyin for treatment skin disorders

ABSTRACT

The present application relates to compositions and methods for treating skin disorders, dry skin, and protection of skin in inflammatory events. The present application more particularly discloses the identification of new genes and metabolic pathways involved in skin disorders, which provide novel targets and approaches for treating said disorders and for screening biologically active compounds. The present invention also provides various products and constructs, such as probes, primers, vectors, recombinant cells, which can be used to implement the above methods. The invention may be used to detect or treat various skin disorders, particularly dry and inflammatory skin disorders, in various subjects, including mammalian subjects, particularly human beings.

The present application relates to compositions and methods for treatingskin disorders. The present application more particularly discloses theidentification of new genes and metabolic pathways involved inichthyosis and, more generally, skin hydration and protection of skin ininflammatory events, which provide novel targets and approaches fortreating said disorders and for screening biologically active compounds.The present invention also provides various products and constructs,such as probes, primers, vectors and recombinant cells, which can beused to implement the above methods. The invention may be used to detector treat various skin disorders, particularly dry skin disorders andinflammatory events, in various subjects, including mammalian subjects,particularly human beings.

The metabolic pathways from arachidonic acid (“AA”), which lead toleukotrienes, hepoxilins and lipoxins, have been mainly defined by thebiochemical identification of their numerous intermediates and products.

Leukotrienes and lipoxins are potent lipid mediators derived from AA,via activation of 5-lipoxygenase and 15-lipoxygenase enzymes. All ofthem belong to the large eicosanoid family of small lipid messengersgenerated by divergent metabolic pathways from AA, and are implicated inbiological functions as diverse as chemotaxis, vascular permeability,smooth muscle contraction, inflammation, and in the pathophysiology ofvarious inflammatory and hypersensitivity disorders such as asthma.Recent reviews have summarized the present knowledge of theirmetabolism, biological actions, and implication in common and raredisorders.

Another metabolic pathway, which also starts from AA but proceeds viaactivation of 12-lipoxygenase, leads to several trihydroxytetraeneproducts of the hepoxilin family (acronym for hydroxy epoxid with abiological activity in insulin secretion). Hepoxilin and otherintermediates or products act primarily on calcium and potassiumchannels in membranes, which ultimately result in a wide range offunctions including insulin secretion, vascular permeability,vasoconstriction, synaptic transmission, cell volume regulation, andactivation of neutrophils and platelets. However, paradoxically, thispathway has received less attention since its discovery during theeighties.

Based on the identification of several genes implicated in a group ofrare dermatological disorders (15-20), the autosomal recessivecongenital ichthyoses (ARCI), the present inventors now disclose a novelmetabolic pathway leading to hepoxilin with 12(R)-chirality, as well asits unexpected and undisclosed implication in skin disorders. Thepresent application thus discloses novel genes, metabolites, targets andtherapeutic approaches to the treatment or prevention of particular skinconditions.

More particularly, the present invention reports the genomiclocalization and the identification of a gene, named ICHTHYIN, for a newform of non-syndromic autosomal recessive congenital ichthyosis (ARCI).Six homozygous mutations, including one nonsense and five missensemutations, were identified in said gene in 23 patients from 14consanguineous families from Algeria, Colombia, Syria and Turkey.ICHTHYIN encodes a protein with several transmembrane domains whichbelongs to a new family of proteins localized in membranes (PFAM:DUF803), with weak homologies to both transporters and G-protein coupledreceptors (GPCR).

Based on the results presented here, it is postulated that ICHTHYIN isthe key membrane receptor for 12(R)-A3 trioxilin from the hepoxilinpathway. For the first time, the present application discloses theimplication of the 12(R)-hepoxilin metabolic pathway in skin disorders.The present inventors also propose, for the first time, a function ofparticular genes in the 12(R)-hepoxilin metabolic pathway.

A particular object of this invention thus resides in a method ofdetecting, diagnosing or characterizing the presence of, orpredisposition to a skin disorder in a subject, comprising assessing, ina biological sample from said subject, the presence of a geneticalteration in the ICHTHYIN gene or corresponding protein, the presenceof a genetic alteration in said gene or protein being indicative of thepresence of or predisposition to a skin disorder in said subject.

As will be disclosed further below, the genetic alteration may be amutation, a deletion, an insertion, a splice site mutation, aninversion, an addition and/or a substitution of one or more residues insaid gene or encoded protein, typically a mutation. The biologicalsample may be any tissue, cell or fluid that contains an ICHTHYIN geneor polypeptide, preferably a sample comprising genomic DNA from thesubject.

A further aspect of this invention resides in nucleic acid probes andprimers that can specifically hybridise to or amplify all or adistinctive part of the ICHTHYIN gene.

A further aspect of this invention is a method of amplifying an ICHTHYINgene, the method comprising:

-   -   (i) providing a biological sample containing an ICHTHYIN gene or        a portion thereof,    -   (ii) contacting said sample with (a pair of) primers as defined        above, and    -   (iii) amplifying the ICHTHYIN gene or mRNA.

Another object of this invention is a method of selecting or identifyingbiologically active compounds, comprising contacting a candidatecompound with an ICHTHYIN protein or a related receptor, and determiningwhether said compound binds to or modulate the activity of said protein.In a particular embodiment, the method comprises contacting the testcompound with a recombinant host cell expressing an ICHTHYIN protein ora related protein, and selecting the compounds that bind said protein ormodulate its activity.

A further object of this invention is a recombinant host cell comprisinga genetic construct encoding an ICHTHYIN protein or a related protein,said cell expressing said protein as a membrane protein. The recombinantcell of this invention may be a prokaryotic or eukaryotic cell. It maybe a primary cell or an established cell line, of various origins.

Still a further aspect of this invention resides in an antibody thatspecifically binds an ICHTHYIN protein or a related protein.

An other object of this invention resides in the use of a compoundselected from a metabolite of the 12(R)-hepoxilin pathway or an analogthereof (for instance, an ester derivative, an amide derivativethereof), for the manufacture of a medication (or medicament) for thetreatment of a skin disorder, as well as in a corresponding method oftreatment. The compound may be selected, for instance, from 12(R)-TXA3,12(R)-HXA3, 12(R)-HXA3-C, 12(R)-HXA3-D and 12(R)-HXA3-E, as well as anyligand of an ICHTHYIN protein, particularly 12(R)-TXA3 or an agonist orsynthetic analog thereof. Those compounds may be prepared in vitro fromarachidonic acid using native or recombinant12R-lipoxygenase encoded byhuman ALOX12B gene, 12R-epoxide isomerase encoded by human ALOXE3 gene,an epoxide hydrolase, or a glutathione S-transferase, a glutamyltranspeptidase, a dipeptidase, or combinations of those enzymes (FIG.5). Synthetic methods for the preparation of racemic HXA3 have also beenreported (39-40).

An other object of this invention is a composition comprising ametabolite of the 12(R)-hepoxilin pathway, or an analog thereof, and apharmaceutically acceptable carrier, preferably for topical application.

Still another object of this invention are metabolites of the12R-hepoxilin pathway such as 12(R)-TXA3, 12(R)-HXA3-C, 12(R)-HXA3-D and12(R)-HXA3-E, and their ester or amide derivatives, usefull for thetreatment of dry skin.

The invention may be used to detect, diagnose, classify, treat(including prevention of) a skin disorder in a subject, particularly adry skin disorder, notably dermatosis, such as atopic skin, contactdermatitis, eczema, psoriasis, or a keratinization disorder such as anichthyosis and a palmoplantar keratoderma, or to protect skin ininflammatory events; or to screen, identify, select or produce compoundsfor use in treating or preventing a skin disorder.

Definitions

Dry skin disorders: Within the context of the present invention, a “dryskin” disorder refers generally to any pathology associated with orresulting from an impaired skin barrier function and/or with epidermisdryness. Dryness and skin barrier disorders are not a single entity, butare characterized by differences in chemistry and morphology in theepidermis. (38). Specific examples of such diseases or disorders includeatopic skin, contact dermatitis, eczema, psoriasis and keratinisationdisorders such as ichthyosis. Skin disorders also include skininflammation as well as skin affections resulting from various type ofaggressions, including chemical (acid, sodium hydroxide, etc.), physical(e.g., X-ray, UV, etc.), or biological (e.g., infections) aggressions.ICHTHYIN gene: Within the context of the present invention, the termICHTHYIN gene refers to a nucleic acid molecule having a sequence asdisclosed in FIG. 4 (SEQ ID No: 1), encoding a receptor for a ligand ofthe hepoxilin metabolic pathway, as well as any portion thereof,naturally occurring variant thereof, such as variants resulting frompolymorphisms, splicings, etc., and orthologs thereof. The term“ICHTHYIN gene” applies to coding and non-coding parts of the genomicDNA or RNA, such as promoter, poly A, intronic sequences, etc. The termgene also includes the coding region, in the form of genomic DNA, cDNA,RNA (pre-rRNA, messenger RNA, etc), etc. or any synthetic nucleic acidcomprising all or part of the sequence thereof. Synthetic nucleic acidincludes cDNA, prepared from RNAs, and containing at least a portion ofa sequence of the ICHTHYIN gene as for example one or more introns or aportion containing one or more mutations. Those nucleic acids can beisolated from known banks by hybridization techniques under stringentconditions at best. They can also be genetically or chemicallysynthesized.

The sequences of wild type human ICHTHYIN gene is available in theliterature under the following accession numbers : LocusID LOC348938;mRNA AK026158 (GI:10438919); mRNA XM_(—)371777 (GI:42657191). Thesequence of mouse ICHTHYIN gene may be found as Mus musculus RIKEN cDNA9530066K23Rik, LocusID 214112; mRNA NM_(—)172524 (GI:27369725) identicalto AK035561 (GI:26330753). The sequence of rat ICHTHYIN gene may befound at LocusID LOC303070; mRNA XM_(—)220330.2 (GI:34870677).

A portion or part of a gene means at least 3 nucleotides, preferably atleast 9 nucleotides, even more preferably at least 15 nucleotides, andcan contain as many as 1000 nucleotides or more. Such a portion can beobtained by any technique well known in the art, e.g., enzymaticcleavage, chemical synthesis or a combination thereof. A distinctiveportion of an ICHTHYIN gene refers to a fragment of at least 5,preferably at least 8 consecutive nucleotides that are specific for asequence as disclosed above. A distinctive portion may also contain agenetic alteration, as disclosed below.

ICHTHYIN protein: Any polypeptide encoded by an ICHTHYIN gene as definedabove. As a specific example, the sequence of wild type human ICHTHYINprotein is available in the literature under the following accessionnumber: XP_(—)371777 (GI:41146935) (SEQ ID No 2), the mouse protein isavailable as NP_(—)766112 (27369726), and the rat protein is availableas XP_(—)220330.1; XP_(—)220330.2 (GI:34870678).

ICHTHYIN related protein: ICHTHYIN related proteins include a group ofreceptors, which have now been identified by the present inventors asbeing involved in arachidonic acid metabolic pathway, which include i)NIPA1 (Non-Imprinted in Prader-Willi/Angelman syndrome 1); ii) NIPA2(DUF803.0); LocusID 81614; mRNA NM_(—)030922 or BC011775 (GI:33989168);iii) LOC152519 (DUF803.3) iv) FLJ13955 (DUF803.1) LocusID 79815;NM_(—)024759 (GI:13376096) identical to AK024017 (GI:10436266); and v)dJ462023.2 (DUF803.4). BLAST analysis identified the above genes ashuman paralogs of ICHTHYIN. Among these genes, the first three are themost closely related to ICHTHYIN, with up to 57% homology. The term“ICHTHYIN related proteins” includes the above listed proteins, in theirwild-type form, as well as naturally occurring variants thereof,resulting for instance from polymorphism, splicing, etc. The amino acidand nucleotide sequences of these members are available in theliterature. The present invention now demonstrates that these proteinshave very significant sequence homology and represent a new family ofreceptors involved in arachidonic acid metabolic pathway.

Identification of a Novel Gene and Metabolic Pathway Involved in DrySkin Disorders

The clinically and genetically heterogeneous group of autosomalrecessive congenital ichthyoses (ARCI) is characterized by generalizeddesquamation of the skin, usually with erythema (1-3). Most of thepatients are born as collodion babies. The disease may progress to twomain clinical forms, either lamellar ichthyosis (LI) or non-bullouscongenital ichthyosiform erythroderma (NCIE). The estimated incidence isbetween one in 300,000 to 500,000 for both forms. LI differs from NCIEin the characteristics of the scales, which are large, adherent, darkand pigmented, and in the absence of skin erythema. In NCIE, the scalesare fine and white on an erythematous background, although they arelarger and grayish on the limbs (3). Overlapping phenotypes have beendescribed, and may depend on age of the patient, and the region of thebody. LI is characterized histologically by orthohyperkeratosis and mildfocal parakeratosis. Hyperkeratosis associated with an increase instratum corneum thickness, a normal or prominent granular layer, andincreased mitoses suggest a hyperproliferative epidermal defect in NCIE(3). Prominent dermal flood vessels and an upper lymphocytic infiltratemay explain the erythroderma. The terminal differentiation of theepidermis is disturbed in both forms, leading to a reduced barrierfunction and defects in the stratum corneum lipid composition (2-5). LIis considered to be a retention ichthyosis, in comparison with NCIE,which is more a hyperproliferative disease (5, 6).

ARCI has been shown to be genetically heterogeneous. Five genes havebeen localized: LI1 (MIM 242300) on chromosome 14q11; LI2 (MIM 601277)on chromosome 2q33-35; LI3 (MIM 604777) on chromosome 19p12-q12;non-lamellar, non-erythrodermic congenital ichthyosis NNCI 19p13.2-p13.1(MIM 604781); and LI5 (MIM 606545), also known as NCIE1 (MIM 242100) onchromosome 17p13 (7-11). Four of these genes have been identified todate: transglutaminase 1 (TGM1) for LI1 (12, 13), two lipoxygenases(ALOXE3 and ALOX12B) for LI5/NCIE1 (14), and ABCA12 for LI3 (15).Another gene, CGI-58 or ABHD5 has been shown to underlie a syndromicform of NCIE2 (MIM 242100) called Chanarin-Dorfinan syndrome (16).

A genome-wide scan of two large consanguineous families with three andfour affected children respectively allowed us to localize a gene onchromosome 5q33. Twelve additional consanguineous families were thenfound to have ichthyosis linked to chromosome 5q33. By microsatellitegenotyping the inventors refined the linkage interval to 2.3 Mb based onrecombination events. Haplotype analysis reduced the interval to 1 Mb.Twelve candidate genes were excluded by sequencing, before the inventorsidentified mutations in a new gene, that the inventors name ICHTHYIN.

The identification of mutations in a new gene with several TM domains inpatients affected by ARCI is a landmark in the analysis of thesedisorders, for which both low incidence and wide clinical and geneticheterogeneity have made it difficult to develop a classification schemeusing clinical (2, 3, 6), biochemical (5, 6, 22), and ultrastructuralcriteria (23). It supports a metabolic origin of ARCI, which wassuggested after two genes from the lipoxygenase family (ALOX12B andALOXE3) were found to be mutated in this disorder (14). This finding wasextended by the identification of ALOXE3 as a hydroperoxide isomerase(24). The phenotype of this ARCI caused by mutations in ICHTHYIN issimilar to that previously described for other ARCIs, in which themajority of patients present a NCIE phenotype. Not all ARCI patients areborn as collodion babies; this was the case in 9 patients from fourAlgerian and two Turkish families.

One of the main difficulties in the present work was the identificationof the gene, which necessitated the sequencing of all the genes presentin the refined interval, and a search for new genes, which weresuspected only by RNA expression. A precise characterization of thestructure of this gene by RT-PCR, 5′ and 3′RACE was obviously required.

With its 7-9 TM domains, ICHTHYIN is clearly membrane-associated. Basedon the results presented here, including the presence of mutationscorrelated to skin disorders, the expression profile and the geneticorganization of certain metabolic pathways, it is submitted thatICHTHYIN is a membrane receptor of the hepoxilin pathway, moreparticularly a receptor for a ligand from the hepoxilin pathway withR-chirality, more precisely the receptor for 12(R)-trioxilin A3 (12(R)-TXA3). This invention is landmark and provides the first descriptionof a receptor for a hepoxilin metabolite with R-chirality, and a key ofthe correlation between the 12(R)-hepoxilin pathway and skin disorders.

Furthermore, the present invention now discloses that other genes foundto be mutated in ichthyoses: the CGI-58 protein or abhydrolase 5 (12,13, 16) and the transporter ABCA12 (15), also belong to the hepoxilinmetabolic pathway, in which membrane arachidonic acid is transformed tohepoxilin, trioxilin and derivatives of R-chirality (25, 26).

The identification of this novel metabolic pathway, including theICHTHYIN protein, offers strong opportunities to provide simpletreatments to patients. In particular, ICHTHYIN, as well as relatedproteins, is easily accessible and enables the design and the synthesisof analogues, agonists and antagonists of the ligand, with highspecificity and sensitivity in the ligand-receptor interactions.

Methods of Diagnosing Skin Disorders

The present invention discloses that the ICHTHYIN gene or encodedpolypeptide is genetically altered in patients having (or at risk ofdeveloping) skin disorders. This gene and polypeptide thus representvaluable biomarkers, suitable for monitoring predisposition, presence orprogression of a skin disorder in a subject.

In this respect, a particular object of this invention resides in amethod of detecting, diagnosing or characterizing the presence of, orpredisposition to a skin disorder in a subject, comprising assessing, ina biological sample from said subject, the presence of a geneticalteration in the ICHTHYIN gene or corresponding protein, the presenceof a genetic alteration in said gene or protein being indicative of thepresence of or predisposition to a skin disorder in said subject.

The alteration in the ICHTHYIN gene or polypeptide may be any geneticalteration, such as a mutation, a deletion, an inversion, an insertion,a splice site mutation, an addition and/or a substitution of one or moreresidues in said gene or encoded polypeptide. Preferred geneticalterations are mutations in a coding or non-coding region, particularlymutations in a coding region leading to a change in amino acid sequencein the encoded polypeptide, e.g., an amino acid substitution, aframeshift and/or a truncated polypeptide sequence. Specific examples ofgenetic alterations in the ICHTHYIN gene are disclosed in Table 1, andinclude a nonsense mutation and five missense mutations: 247C→T (R83X)and 239→T (G80V) in exon 2; 341C→A (A114N) in exon 4, 437C→T (S146F) and523C→G (H175N) in exon 5; and 703G→A (G235R) in exon 6. The numberingcorresponds to the coding sequence only, starting with 1 from the A ofthe initiating ATG codon (corresponding to methionin) (see FIG. 4, boldcharacters). By reference to the sequence of accession numberXM_(—)371777, which is the reference sequence of NCBI, 1=A of the ATGcorresponds to bp 106, and goes until bp 1320=A of the TGA(corresponding to a stop codon). By reference to the sequence ofaccession number AK026158, which is the sequenced cDNA, 1=A of the ATGcorresponds to bp 107, and goes until bp 1321=A of the TGA(corresponding to a stop codon). Numbering of amino acid residues refersto XP_(—)371777.

The presently identified mutations are situated in parts of the genethat are highly conserved between mice, rats and humans. None of thesesequence variations were found in 100 normal chromosomes from aMediterranean control population.

The methods comprise, typically, detection of the presence of a mutationin an ICHTHYIN gene or polypeptide in a biological sample from asubject. The biological sample comprises any material such as cells, atissue, an organ, a fluid, etc or fractions thereof, which containnucleic acids or polypeptides. Specific examples of such biologicalsamples include white blood cells or fluids, such as blood, plasma orurine, biopsies, skin and mucosa, which may possibly be obtained bynon-invasive methods or from tissue collections, if necessary.Furthermore, since the ICHTHYIN gene is found within the cells, tissuesor fluids mentioned above, the sample is usually treated to render thegene available for detection or analysis. Treatment may comprise anyconventional fixation technique, cell lysis (mechanical or chemical orphysical), or any other conventional method used in immuno-histology orbiology, for instance.

In a first variant, the present invention, provides a method ofdetecting the presence of an altered ICHTHYIN polypeptide. This can bedetermined by any suitable technique known to the skilled artisan,including by immuno-assay (ELISA, EIA, RIA, etc.). This can be madeusing any affinity reagent specific for an ICHTHYIN polypeptide, morepreferably any antibody or fragment or derivative thereof, particularlyany affinity reagent specific for an altered ICHTHYIN polypeptide. In aparticular embodiment, the ICHTHYIN polypeptide is detected with ananti-ICHTHYIN antibody (or a fragment thereof), more preferably amonoclonal antibody, as described above. The antibody (or affinityreagent) may be labeled by any suitable method (radioactivity,fluorescence, enzymatic, chemical, etc). Alternatively,ICHTHYIN-antibody immune complexes may be revealed (and/or quantified)using a second reagent (e.g., antibody), labelled, that binds to theanti-ICHTHYIN antibody, for instance.

In a second variant of the present invention, the presence of an alteredICHTHYIN gene is detected. This can be done using various techniques, asdescribed below.

In a particular embodiment, the method comprises the characterization ofall or part of an ICHTHYIN gene in the sample and the comparison of saidgene to the wild-type ICHTHYIN gene. Comparison can be made by (partial)sequencing, gel migration, hybridization, etc.

In another particular embodiment, the method comprises detecting all orpart of an ICHTHYIN gene in the sample by selective hybridisation to aspecific nucleic acid probe. Another method of the invention comprisesthe amplification of an ICHTHYIN gene or a portion thereof, and thedetermination of the presence of an alteration in the amplificationproduct.

In particular embodiments, ICHTHYIN gene alterations are assessed byquantitative RT-PCR, LCR (Ligase Chain Reaction), TMA (TranscriptionMediated Amplification), PCE (an enzyme amplified immunoassay) or bDNA(branched DNA signal amplification) assays.

In a particular embodiment, an ICHTHYIN gene alteration is determined byin vitro or ex vivo cDNA synthesis, (PCR) amplification withICHTHYIN-specific oligonucleotide primers, and analysis of PCR products.

RT-PCR amplification of an ICHTHYIN mRNAs or gene may be performed usingany pair of ICHTHYIN-specific primers. In particular any primers can bedesigned by the skilled artisan, such as any fragment of an ICHTHYINgene, for use in the amplification step and especially a pair of primerscomprising a forward sequence and a reverse sequence wherein saidprimers of said pair hybridize with a region of an ICHTHYIN gene (orflanking an ICHTHYIN gene) and allow amplification of at least a portionof the ICHTHYIN gene or of a portion of the ICHTHYIN gene containing agenetic alteration. In a particular embodiment, the ICHTHYIN cDNA can beprepared by using current protocol with a first primer set: RT4A 5′GCCACGCGGGGGACAAT 3′ (SEQ ID No 3) and AK1M 5′ CCTGCAGGCACTGATGTAAA 3′(SEQ ID No 4); and a second primer set selected from the groupconsisting of: RT2A 5′ GACAAGTCGCGGCCACCT 3′ (SEQ ID No 5) and AK3M 5′CTCAAGAAAAGAGAGCCCATTG 3′; (SEQ ID No 6) RT3A 5′ ACAAGTCGCGGCCACCTG 3′(SEQ ID No 7) and RT4M 5′ CAGGAAGTTCTGCCACCATTG 3′; (SEQ ID No 8) andRT2A 5′ GACAAGTCGCGGCCACCT 3′ (SEQ ID No 7) and AK1M 5′CCTGCAGGCACTGATGTAAA 3′. (SEQ ID No 6)

Primers of this invention more preferably contain less than about 50nucleotides even more preferably less than 30 nucleotides, typicallyless than about 25 or 20 nucleotides. Also, preferred primers usuallycontain at least 5, preferably at least 8 nucleotides, to ensurespecificity. The sequence of the primer can be prepared based on thesequence of an ICHTHYIN gene, to allow full complementarity therewith,preferably. Specific examples of primers of this invention are providedin Table 2.

Nucleic acid probes of this invention comprise (or specificallyhybridise to) all or a distinctive part of the nucleic acid sequence ofthe ICHTHYIN gene, preferably at least a distinctive part thereof, i.e.,a portion comprising at least one of the above-mentioned mutations. Theprobes may comprise between 8 and 1000 nucleotides, or even more (e.g.,the entire sequence of the gene). The probes are most preferably singlestranded. The probes may be labeled using any known technique such asradioactivity, fluorescence, enzymatic, chemical, etc. This labeling canuse for example Phosphor 32, biotin (16-dUTP), digoxygenin (11-dUTP).

It should be understood that the present invention shall not be bound orlimited by particular detection or labeling techniques, which canessentially be applied to the ICHTHYIN gene using ordinary skills.

The invention also relates to kits for implementing the above methodscomprising at least a primer or a probe specific for an ICHTHYIN geneand, optionally, reagents for a nucleic acid amplification orhybridization reaction. The reagents may include antibodies, probes,primers, devices, supports, etc.

The invention also relates to a nucleic acid comprising a mutatedICHTHYIN gene sequence or a distinctive portion thereof.

As shown in the example, the invention now demonstrates a correlationbetween the presence of an alteration in an ICHTHYIN gene in a subjectand the presence, development or predisposition to a skin disorder.

Methods of Screening Biologically Active Compounds

In a further aspect, the present invention also relates to the use of anICHTHYIN gene or polypeptide as a target for screening biologicallyactive agents, particularly compounds active on dry skin disordersand/or on the hepoxillin pathway.

A particular object of this invention lies in methods of selecting oridentifying biologically active compounds, comprising contacting acandidate compound with an ICHTHYIN protein or a related protein anddetermining whether said compound binds to or modulate the activity ofsaid protein. Binding may be determined by any technique known per se inthe art, including ligand displacement, competition assays, directbinding using labeled compounds, immuno-precipitation, gel migration,etc. Modulation of the activity may be determined by assessing anyconformational change, or by determining or measuring any secondarysignal mediated by ICHTHYIN or said related protein. The method may becarried out using ICHTHYIN or related protein or a fragment of thereof(for instance the ligand-binding domain), which may be in isolated form,immobilized on a support, expressed in a lipid membrane or by an intactcell.

In a particular embodiment, the method comprises contacting a testcompound with a recombinant host cell expressing an ICHTHYIN (orrelated) protein or a fragment thereof, typically a ligand-bindingfragment thereof, and selecting the compounds that bind said protein ormodulates its activity. Such recombinant cells, which also representparticular objects of the present invention, may be any cell comprisinga genetic construct encoding an ICHTHYIN (or related) protein, said cellexpressing said protein as a membrane protein. The recombinant cell maybe a prokaryotic cell or a eukaryotic cell. Examples of prokaryoticcells include bacterial cells, particularly E. coli. Eukaryotic cellsinclude yeast cells (e.g., saccharomyces, Kluyveromyces, etc), mammaliancells, plant cells, insect cells, etc. Particular examples of mammaliancells include primary or established cell cultures from various species,including rodents, canine, equine, bovine, ovine, human, etc, and fromvarious tissue cell type (e.g., keratinocytes, fibroblasts, hepatocytes,muscle cells, nervous cells, kidney cells, ovary cells, etc).

The recombinant cells may be prepared by conventional recombinanttechniques, e.g., by introduction into a selected cell type of a geneticconstruct encoding an ICHTHYIN (or related) protein, under conditionsallowing expression of said protein as a membrane protein. The geneticconstruct may be any plasmid, cosmid, artificial chromosome, virus,phage, episome, etc, which may be prepared according to techniques knownin the art. The construct typically comprises, upstream from the codingsequence, a promoter region that causes expression of ICHTHYIN in theselected cell. The construct may also include an origin of replication,or a marker gene, or a homologous region (allowing site-specificintegration into the cell's genome), an integrase, etc. The constructmay be introduced into the cells (or their ancestors) by techniquesknown per se in the art, such as electroporation, calcium-phosphateprecipitation, conjugation, naked DNA transfer, transfection, infection,etc. Introduction may be performed in the presence of facilitatingagents, such as liposomes, cationic lipids, polymers, etc. Therecombinant cells may be selected and cultivated under conventionalconditions. Cell surface expression of ICHTHYIN or related protein maybe verified by any binding assay, for instance in the presence of anantibody or ligand.

In this regard, a particular object of this invention is a genetic ornucleic acid construct comprising a nucleic acid sequence encoding anICHTHYIN protein, under the control of a promoter, preferably aheterologous promoter. The heterologous promoter may be any promoterthat does not control ICHTHYIN expression in a naturally occurring cell.Examples of such promoters include viral promoters (e.g., CMV, LTR, TK,SV40, etc), cell promoters (PGK, HAS, etc), bacterial promoters (Trp,Lac, etc), yeast promoters, as well as any artificial or chimericpromoter sequence. The construct may be incorporated into a vector asdescribed above.

A particular aspect of this invention resides in a method of selectingor identifying biologically active compounds, comprising contacting acandidate compound with a recombinant host cell expressing a receptorselected from ICHTHYIN, NIPA1, NIPA2, LOC152519, FLJ13955 anddJ462023.2, or a ligand-binding fragment or sub-unit thereof, andselecting the compounds that bind said protein or modulate its activity.

An other particular aspect of this invention resides in a method ofselecting or identifying a compound that modulates the arachidonic acidmetabolism, comprising contacting a candidate compound with arecombinant host cell expressing a receptor selected from ICHTHYIN,NIPA1, NIPA2, LOC152519, FLJ13955 and dJ462023.2, or a ligand-bindingfragment or sub-unit thereof, and selecting the compounds that bind saidprotein or modulate its activity.

The ligand-binding domain of ICHTHYIN is located in the NH2-terminalregion of the protein, between amino acid residues 1 and 60.Accordingly, in the above selection methods, it is preferred to use aprotein or polypeptide comprising at least amino acid residues 1 to 60of an ICHTHYIN protein.

Preferred compounds of this invention are selected for their ability tobind the above proteins and to behave as agonists of the correspondingligands.

Other methods of this invention comprise contacting a candidate moleculewith a gene encoding said proteins, and selecting the molecules thatbind to said gene or modulate the expression of said proteins.Particular molecules are those, which stimulate expression of saidgenes, particularly those that stimulate the transcriptional promotersof said genes.

Within the context of this invention, various candidate compounds may betested in parallel, using different assay formats (microtitration plate,etc). The compound may be contacted with the cells for a sufficientperiod of time to allow binding to occur, and the binding or activitymay be assessed as disclosed above. The invention is particularly suitedto screen agonists of the ICHTHYIN receptor, or activators thereof.

Antibodies

Another aspect of this invention relates to an antibody that binds anICHTHYIN polypeptide or a related protein. The antibody may be apolyclonal or a monoclonal antibody. Furthermore, the term antibody alsoincludes fragments and derivatives thereof, in particular fragments andderivatives of said monoclonal or polyclonal antibodies havingsubstantially the same antigenic specificity. These include antibodyfragments (e.g., Fab, Fab′2, CDRs, etc), humanized antibodies,poly-functional antibodies, Single Chain antibodies (ScFv), etc. Thesemay be produced according to conventional methods, includingimmunization of an animal and collection of serum (polyclonal) or spleencells (to produce hybridomas by fusion with appropriate cell lines).

To produce polyclonal antibodies from various species, the antigen isgenerally combined with an adjuvant (e.g., Freund's adjuvant) andadministered to an animal, typically by sub-cutaneous injection.Repeated injections may be performed. Blood samples are collected andimmunoglobulins or serum are separated. Monoclonal antibodies may beproduced from various species as described for instance in Harlow et al(Antibodies: A laboratory Manual, CSH Press, 1988). Briefly, thesemethods comprise immunizing an animal with the antigen, followed by arecovery of spleen cells, which are then fused with immortalized cells,such as myeloma cells. The resulting hybridomas produce the monoclonalantibodies and can be selected by limit dilutions to isolate individualclones. Antibodies may also be produced by selection of combinatoriallibraries of immunoglobulins, as disclosed for instance in Ward et al(Nature 341 (1989) 544).

Fab or F(ab′)2 fragments may be produced by protease digestion,according to conventional techniques. Humanized antibodies can beprepared as previously described (Jones 1986; Riechmann 1988).

Preferred antibodies of this invention are prepared by immunization witha fragment of an ICHTHYIN polypeptide or related protein, preferablywith an immunogenic fragment thereof, e.g., a fragment comprising atleast an epitope, preferably of at least 5 amino acids. In a preferredembodiment, the fragment of ICHTHYIN is the C-17-K peptide (SEQ ID No9): Cys Asp Asn Ile Glu Leu Ala Ser Thr Ser Ser Pro Glu Glu Lys Pro Lys.

Other preferred antibodies are monoclonal antibodies that specificallybind an altered ICHTHYIN polypeptide, particularly a mutated ICHTHYINpolypeptide.

The antibodies may be coupled to heterologous moieties, such as toxins,labels, drugs or other therapeutic agents, covalently or not, eitherdirectly or through the use of coupling agents or linkers.

The antibodies of this invention have various applications, includingtherapeutic, diagnostic, purification, detection, prophylactic, etc. Invitro, they can be used as screening agents or to purify the antigenfrom biological samples. They can also be used to detect or quantify thepresence (or amounts) of an ICHTHYIN polypeptide in a sample collectedfrom a subject, typically a blood sample from a mammalian, specificallya human subject. Antibodies of this invention may also be used asagonists or antagonists of the ICHTHYIN receptor, particularly in atherapeutic context.

Novel Compounds and Methods for Treating Skin Disorders

As mentioned above, the present invention discloses a novel metabolicpathway leading to hepoxilin with (R)-chirality, as well as itsunexpected and undisclosed implication in skin disorders. The presentapplication thus allows the design of novel therapeutic approaches tothe treatment or prevention of particular skin conditions, usingcompounds from this metabolic pathway, as well as analogs or agoniststhereof, or compounds that regulate the expression of genes involved inthis metabolic pathway (see FIG. 5).

A particular object of this invention more specifically resides in theuse of a compound selected from a metabolite of the 12(R)-hepoxilinpathway or an analog thereof (for instance, an ester derivative, anamide derivative), for the manufacture of a medication for the treatmentof a skin disorder or a dry skin condition. The compound is preferablyselected from the group consisting of 12(R)-TXA3, 12(R)-HXA3,12(R)-HXA3-C, 12(R)-HXA3-D and 12(R)-HXA3-E (FIG. 3).

-   12-(R)-TXA-3 designates 12-(R)-Trioxilin A-3, i.e.,    8,11R,12R-trihydroxyeicosa-(5Z,9E,14Z)-trienoïc acid (formula    C₂₀H₃₄O₅).-   12-(R)-HXA-3 designates 12-(R)-Hepoxilin A-3, i.e.,    8-(R,S)-hydroxy-(11R,12R)-epoxyeicosa-(5Z,9E,14Z)-trienoïc acid    (formula C₂₀H₃₂O₄).-   12-(R)-HXA-3-C designates 12-(R)-Hepoxilin A-3-C, i.e.,    8-(R,S)-hydroxy-(11S)-glutathionyl-(12R)-hydroxyeicosa-(5Z,9E,14Z)-trienoïc    acid (formula C₃₀H₄₉N₃O₁₀S).-   12-(R)-HXA-3-D designates 12-(R)-Hepoxilin A-3-D, i.e.,    8-(R,S)-hydroxy-(11S)-cysteïnylglycinyl-(12R)-hydroxyeicosa-(5Z,9E,14Z)-trienoïc    acid (formula C₂₅H₄₂N₂O₇S).-   12-(R)-HXA-3-E designates 12-(R)-Hepoxilin A-3-E, i.e.,    8-(R,S)-hydroxy-(11S)-cysteïnyl-(12R)-hydroxyeicosa-(5Z,9E,14Z)-trienoïc    acid (formula C₂₃H₃₉NO₆S).

Those compounds may be prepared in vitro from arachidonic acid usingnative or recombinantl2R-lipoxygenase encoded by human ALOX12B gene,12R-epoxide isomerase encoded by human ALOXE3 gene, an epoxide hydrolase(human soluble or ), or a glutathione S-transferase, a glutamyltranspeptidase, a dipeptidase, or combinations of those enzymes (FIG.5). Synthetic methods for the preparation of racemic HXA3 have also beenreported (39-40).

An analog of the metabolite may be any compound having the same type ofbiological activity. Typically, the analog is a synthetic molecule,having essentially the same core structure, which may contain additionalor alternative substituting groups or chemical functions. Such analogsmay be produced by drug design techniques, which are known in the art.Analogs include substituted or derivatized forms of the metabolitehaving an increased stability, in vivo half-life or which can be moreeasily produced. The term analog particularly encompasses apro-medicament, i.e., a compound that is transformed in vivo into themetabolite. Such analogs may be screened using any one of the abovedisclosed screening methods.

In a more particular embodiment, the compound is a ligand of an ICHTHYINprotein, particularly identified or selected using a screening method asdescribed above.

In a specific, preferred embodiment, the compound is 12(R)-TXA3 or anagonist thereof, i.e., a compound that binds the same receptor andcauses activation thereof.

In this respect, a particular object of this invention resides in theuse of 12(R)-TXA3 or an agonist or synthetic analog thereof, for themanufacture of a medication (or composition) for the treatment of a skindisorder, including dry skin (xerosis) and protection of skin ininflammatory disorders and other aggressions.

The present invention also relates to methods of treating a skindisorder, comprising administering to a subject in need thereof anamount of a metabolite of 12(R)-hepoxilin pathway or an analog thereofeffective for treating said disease. The compounds may be administeredthrough various routes, including oral, systemic and topical routes,particularly by topical administration. The compounds may be formulatedin any appropriate buffer or excipient, such as saline solutions,isotonic buffer, gel, paste, ointment, etc.

In this respect, an other object of this invention is a composition,particularly a cosmetic composition, comprising a metabolite of12(R)-hepoxilin pathway, or an analog thereof, and a pharmaceuticallyacceptable carrier, particularly for topical application.

A particular composition of this invention comprises 12(R)-TXA3 or anagonist or synthetic analog thereof, and a pharmaceutically acceptablecarrier, particularly for topical application.

For topical administration, the composition may be in the form of anointment, gel, cream, soap, foam, salve, spray and the like. Suitableexcipients include any vehicle or agent that is non-toxic in vivo. Fortopical application to the skin, the composition may be in the form ofan aqueous or oily solution, suspension or dispersion, which may beliquid or semi-liquid, such as a lotion, serum, milky preparation, etc.The composition may be in emulsified form, either oil-in-water orwater-in-oil can. It may also be in the form of an anhydrous cream orgel, microparticles, dispersion, etc.

Suitable excipients may include solubilizing agents, stabilizing agents,penetrants, emulsifying agents, surfactants, etc., either alone or incombination(s). The cosmetic composition may also contain additionalagents such as gelling agents, antioxidants, solvents, flavoring agents,preservatives, etc. The respective amounts of these agents may beadjusted by the skilled artisan, typically within the range of 0.01% to15% of the total weight of the composition. Suitable emulsifiers for usein the present invention include, without limitation, glycerol,polysorbate and stearate. Suitable gelling agents include, withoutlimitation, carboxyvinyl and acrylic copolymers, polysaccharides (e.g.,hydroxypropylcellulose), polyacrylamides, clays, aluminium stearates,ethylcellulose and polyethylene. If appropriate, for topicaladministration, a thickening agent such as methyl cellulose may be usedas well.

For oral administration, the compounds may be formulated into anyconventional dosage form, including tablets, capsules, ampoules, etc.For transmucosal or transdermic administration, they may be formulatedin the presence of penetrants, as gels, sprays, patch, etc.

The above methods, uses and compositions can be used to treat skindisorders, either alone or in combination with other agents, includingto reduce the progression, prevent the development, suppress anysymptoms or completely abolish the disease. The compounds may beadministered according to various protocols, which may be adjusted bythe practitioner, including the use of repeated administrations. Typicaldosages may vary for instance from 0,01 to 1000 mg of active agent perdose.

As disclosed above, the skin disorder is selected from a group of dryskin disorders, particularly a dermatosis such as atopic skin, contactdermatitis, eczema, a psoriasis, or a keratinization disorder such as anichthyosis and a palmoplantar keratoderma.

In another aspect, the skin disorder is selected from any skininflammation or aggression, including burnt skin, acid, UV, etc.

The invention also relates, generally, to a pharmaceutical compositioncomprising an agonist or an antagonist of an ICHTHYIN protein (such asan antibody or a fragment thereof, or any synthetic molecule), incombination with a pharmaceutically acceptable vehicle or excipient,particularly for topical administration.

Further aspects and advantages of this invention will be disclosed inthe following examples, which should be considered as illustrative andnot limiting the scope of this application.

LEGENDS TO THE FIGURES

FIG. 1: Pedigrees of families F1 to F14.

Mutations in ICHTHYIN. Sequences are shown in 2 affected patients fromfamilies F4 and F10, one parent, and one normal control.

FIG. 2 : Patient's haplotypes and corresponding mutations. Loss ofhomozygosity is indicated by gray color. Inside the smallest segregatinginterval between markers a083xb9 and D5S378, mutations and commonalleles are in red. Allele number is indicated as 0 when no genotypingresult is available. Paternal and maternal alleles for each affectedchild are presented.

FIG. 3 : Structure of active compounds of this invention.

FIG. 4: Nucleotide sequence of an ICHTHYIN gene coding portion.

FIG. 5: A novel metabolic pathway

EXAMPLES

Materials and Methods

Subjects and Samples

The dermatologists recorded the clinical data and pedigree informationof the families. Blood samples were collected from each participatingfamily member after obtaining written informed consent. DNA extractionfrom peripheral blood leukocytes and establishment of cell lines wereperformed using standard procedures.

Genetic Analysis

Genotyping was carried out using 400 highly polymorphic microsatellitemarkers from the ABI panel (Linkage Mapping Set2, LMS2, AppliedBiosystems) and a MegaBase capillary sequencer for the genome-wide scan.ABI 377 sequencers were used for fine mapping with publicly availablemicrosatellites. Haplotypes were constructed assuming the mostparsimonious linkage phase. Linkage programs were used based on theassumption of autosomal recessive inheritance, full penetrance and adisease frequency of 1/500,000 in the general population. Pairwise LODscores were calculated with the MLINK program of the LINKAGE 5.1 package(36) incorporating consanguineous loops into the pedigree files.

Mutation Screening

Mutation analysis was performed in affected patients and in both parentsin the 14 families, and in supplementary non-affected sibs in cases ofmissing parents. The inventors designed intronic oligonucleotide primersflanking the exons for amplification and sequencing the ICHTHYIN gene(Table 2) using the Primer3 program(http://www-genome.wi.mit.edu/genome_software/other/primer3.html) (37).Sets of PCR conditions were used as indicated in Table 2. The touch-downPCR reaction was performed in a 45 μl volume containing 50 ng of genomicDNA (in 5 μl) with Hot Master™ Taq DNA polymerase (Eppendorf): initialdenaturation step at 95° C. for 5 min, 6 cycles of amplificationconsisting of 40 s at 94° C., 30 s 68° C., and a 30 s elongation step at72° C., followed by 30 cycles of 40 s at 94° C., 30 s at optimalannealing temperature, 30 s at 72° C., and a 5 min terminal elongationstep. One to 2 μl of purified PCR products were added to 0.5 μl of senseor antisense primer (20 μM) and 2 μl of BigDye terminator mix (AppliedBiosystems) in a 15 μl volume. The linear amplification consisted of aninitial 5 min denaturation step at 96° C., 25 cycles of 10 s ofdenaturation at 96° C. and a 4 min annealing/extension step at 56-60° C.The reaction products were purified and sequenced on an AppliedBiosystems Sequencer 3700. The forward or reverse strands from allpatients and controls were sequenced for the entire coding region andthe exon/intron boundaries. The sequences were analysed with the PhredPhrap program on Unix.

Lymphoblastoid, Keratinocyte and Fibroblast Cell Cultures, and RNAExtraction

Lymphoblastoid cell lines were established using standard procedures.Total RNA from lymphocytes was extracted with the RNA-PLUS(Quantum-Appligene) kit following the manufacturer's instructions. Humankeratinocytes and fibroblasts were obtained from skin removed duringroutine plastic surgery of a normal individual. The skin sample wasprocessed for primary keratinocyte culture and cells were grownaccording to the standard procedure described by Invitrogen LifeTechnologies using products from the same company in serum-freekeratinocyte medium supplemented with bovine pituitary extract (25μg/ml) and recombinant epidermal growth factor (0.1 ng/ml). For primaryfibroblast culture the inventors used DMEM (Dulbecco's Modified Eagle'sMedium) with 10% fetal calf serum and 2% L-glutamine. Cultures weregrown for two passages and harvested when they reached 90% confluence.Total RNA was isolated using the QIAamp RNA Mini Protocol for isolationof total RNA from cultured cells (QIAGEN) following the manufacturer'sinstructions. The mRNA was isolated following the Oligotex direct mRNAprotocol as provided by the manufacturer (QIAGEN).

5′-RACE (Rapid Amplification of cDNA Ends) and Cloning of theAmplification Products

5′-RACE was performed with Marathon-Ready cDNA from melanoma, placentaand stomach (Clontech) using Advantage 2 Polymerase Mix (Clontech)following the manufacturer's instructions. The PCR was run with AP1primer and one of the five gene-specific antisense primer which theinventors designed from the cDNA sequence, starting with an initialdenaturation step at 94° C. for 1 min, 30 cycles at 94° C. for 30 s and68° C. for 4 min. To visualize the PCR products, they were loaded on a2% agarose gel.

RT-PCR and Rapid-Scan™ Gene Expression Panel

RT-PCR was performed using the RT-PCR kit (Life Technologies) with oligodT primers to generate the first strand of cDNA. Amplification of cDNAfrom keratinocytes, fibroblasts, placenta and lymphocytes was performedwith four primer pairs (Table 2) covering the entire coding region, the3′UTR and the 5′UTR region.

A gene expression panel including 24 human tissues was tested followingthe manufacturer's instructions (Rapid-Scan, OriGene Technologies) usingprimer pair RT_(—)4 which is specific for the ICHTHYIN transcript.Expression in keratinocytes, fibroblasts, placenta and lymphocytes wasalso tested.

Additional Accession Numbers

Online Mendelian Inheritance in Man (http://www.ncbi.nlm.nih.gov/Omim):Abhydrolase domain containing 5 (ABHD5), previously CGI58, ComparativeGene Identification 58 [CGI58; MIM 604780]; Arachidonate lipoxygenase 3[ALOXE3; MIM 607206]; Arachidonate 12-lipoxygenase, R type [ALOX12B; MIM603741]; ATP-binding cassette, subfamily A, member 12 [ABCA12; MIM607800]; Chanarin-Dorfman syndrome [CDS; MIM 275630]; Ichthyosisnonlamellar and nonerythrodermic congenital [NNCI; MIM 604781]; Lamellarichthyosis [LI; MIM 242300]; Lamellar ichthyosis 1 [LI1; MIM 604777];Lamellar ichthyosis 2 [LI2; MIM 601277]; Lamellar ichthyosis 3 [LI3; MIM604777]; Lamellar ichthyosis 5 [LI5; MIM 606545]; Nonbullousichthyosiform erythroderma [NCIE1, MIM 242100]; Nonbullous ichthyosiformerythroderma [NCIE2, MIM 604780]; Nonimprinted gene in Prader-Willisyndrome/Angelman syndrome chromosome region 1 [NIPAI; MIM 6008145];Nonimprinted gene in Prader-Willi syndrome/Angelman syndrome chromosomeregion 2 [NIPA2; MIM 6008146]; Presenilin 1 [PSEN1; MIM 104311];Transglutaminase 1 [TGM1; MIM 190195]; Spastic paraplegia 6, autosomaldominant [SPG6; MIM 600363].

Results

Clinical Features and Patient Origins

The inventors analyzed 14 families comprising 23 patients (12 females,11 males) and 50 non-affected family members. Individuals for whom DNAwas available are shown in FIG. 1. All families are consanguineous fromfirst cousin marriages, except two families in which the parents are 2ndcousins (F1) and 2nd cousins once removed (F14) (Table 1). Most familieswere from Mediterranean countries (eight from Algeria, four from Turkey,one from Syria), and one was from Colombia.

The majority of patients (60%) were born as collodion babies except forthe patients from four Algerian families (F2, F4, F9, F14) and twoTurkish families (F6, F8) who presented a clinical picture resemblingNCIE. They presented generalized ichthyosis with erythema, fine whitishscaling on the face and trunk, and larger brownish scaling on the neck,buttocks and legs (see FIG. 2). However some of the families showed amore lamellar phenotype. All the patients presented palmoplantarkeratoderma, often yellowish with fissures, and some had clubbing ofnails.

Linkage, Linkage Disequilibrium and Haplotype Analysis

The inventors first localized the ICHTHYIN gene on chromosome 5q33.2-q34by homozygosity mapping in two consanguineous families (F1 and F14) withthree and four affected individuals. A region of homozygosity betweenthe markers D5S410 (AFM191xd8) and D5S422 (AFM211yc7) was confirmed witha few additional microsatellite markers, before the inventors screenedour DNA collection to find more families with ichthyosis linked to thisnew locus. A total of 81 families were analysed; 64 were consanguineous,and 17 were non-consanguineous but had more than one affected familymember. The six known ichthyoses localizations were previously excludedin all of these families using our panel of 30 microsatellite markerscovering the loci on chromosomes 2q33-35, 3p2, 14q11, 17p13, 19p12-q12,and 19p13.2-p13.1 (7-11). In 12 of these families (F2 to F13) linkage ofichthyosis to the new localization was found. The inventors thenperformed fine mapping with a total of 29 microsatellites (FIG. 2) in a15.1 Mb interval between markers AFM336xe9 (D5S2077) and AFMb351xf9(D5S2050). The maximum pairwise LOD score at Θ=0.00 for D5S378 was16.38. A co-segregating region of 1 Mb was homozygous in all patients,defined by recombination events with loss of homozygosity in one patientfrom family 5 for the centromeric marker AFMa083xb9 (D5S2112), and in apatient from family 14 for the telomeric marker AFMb343xe9 (D5S2049).The results of genotyping are presented in FIG. 3. Patients from sevenfamilies (F5 to F11) shared a haplotype for 2 to 5 markers (2-7-4-4-3)inside the interval including the gene; these seven families, which haddifferent geographical origins (Algeria, Colombia and Turkey), werelater shown to carry the same missense mutation. Three other familiesfrom Algeria (F2 to F4) exhibited the same nonsense mutation, but onlytwo of them shared the same haplotype.

Exclusion of Candidate Genes and Identification of Mutations in a NovelGene ICHTHYIN

Five genes were sequenced in the initial 2.3 Mb interval between themarkers UT2159 and AFMb343xe9 (LOC91937, HAVCR1, HAVCR2, CRSP9,MGC26988) and all 7 known genes in the refined 1 Mb interval (CYFIP2,PRO133 ADAM19, SOX30, FLJ20546, FLJ38273 and ENTH). No mutations werefound in the coding regions or exon-intron boundaries of these 12 genes.The inventors started therefore to analyze the human mRNAs and ESTs fromGenBank between the markers AFMa083xb9 (D5S212) and AFMb343xe9(D5S2049). One mRNA (AK026158, AF131815) was of particular interestbecause it was highly expressed in epidermal tissues, including skin andkeratinocytes. Sequencing of the corresponding gene revealed 6 differentmutations.

Structure of the ICHTHYIN Gene and its cDNA

A new predicted sequence (XM_(—)371777) of 3077 bp was proposedcorresponding to a cDNA (AK026158, FLJ22505) of 3094 bp (including thepoly-A tail), which codes for a protein of 404 amino acids. BLASTanalysis between this mRNA and the BAC sequence AC008676 supported theexistence of six exons as described in other public databases. Theinventors performed 5′RACE with 5 different primers and subcloned someof these 5′RACE products, but the inventors failed to identify anyelongation of the 5′ end. The sequence was checked by overlappingRT-PCR, and the products were sequenced and compared with the sequencesfrom public databases. BLAST analysis revealed one mouse (NM_(—)172524)and one rat mRNA ortholog (XM_(—)220330) showing homologies of 83% and84% for the nucleotide sequence, and of 74% and 75% for the proteinsequence respectively. The mouse mRNA of 3299 bp is reported to befull-length whereas the rat mRNA is only 1272 bp long. Multiplenucleotide alignments (http://prodes.toulouse.inra.fr/multalin/) oforthologs from human, mouse and rat showed a highly conserved sequencewith a homology of 92%.

Mutation Analysis of the ICHTHYIN Gene

Sequencing of the six exons and of the exon-intron boundaries of theICHTHYIN gene revealed six different homozygous mutations in the 14consanguineous families (Table 1): one was a nonsense mutation and fivewere missense mutations: 247C→T (R83X) and 239G→T (G80V) in exon 2;341C→A (A114N) in exon 4, 437C→T (S146F) and 523C→G (H175N) in exon 5;and 703G→A (G235R) in exon 6. The mutations were all situated in partsof the gene that are highly conserved between mice, rats and humans.None of these sequence variations were found in 100 normal chromosomesfrom a Mediterranean control population.

Expression Analysis

The inventors analyzed tissue expression by ICHTHYIN-specific RT-PCRwith primer pairs RT_(—)4 (Table 2) for a 1111 bp fragment using theRAPID-SCAN™ gene expression panel and RNAs from cultured keratinocytes,fibroblasts, placenta and lymphocytes (data not shown). The ICHTHYINtranscript was found to be expressed in most tissues tested; it washighly expressed in brain, lung, stomach, skin and leucocytes, and waspresent at a lower level in the other tissues, with the exception ofliver, thyroid and fetal brain in which no expression was detectable.Strong expression of ICHTHYIN transcripts was observed in culturedkeratinocytes from normal skin biopsies; expression was weaker incultured fibroblasts from the same skin biopsies, in placenta and inlymphocytes.

Sequence Analysis of the ICHTHYIN Protein and Identification ofConserved Residues

The sequence of 404 amino acids corresponds to a protein with acalculated molecular weight of 44 kDa. This sequence was submitted tothe topology prediction programs for sequence signals, transmembrane(TM) domains and protein orientation available through the ExPASy server(www.expasy.org), to protein analyzing tools from the Biology WorkBench(http://biowb.sdsc.edu/) and to homology searches through proteindatabases (http://www.ncbi.nlm.nih.gov). The protein is predicted tolocalize in the plasma membrane, but does not possess a signal sequence.The protein was determined to contain 7-9 TM domains, using the PSORTII,SMART and BPROMPT programs and the Dense Alignment Surface (DAS) method.This structure defines a new family of proteins (PFAM: DUF803). Analysisusing the ProtFun2.1 server (http://www.cbs.dtu.dk/services/ProtFun/)classifies ICHTHYIN in finctional and gene ontology categories undertransport and binding activities.

A New Family of Proteins with Several TM Domains

BLAST analysis identified five human paralogs of ICHTHYIN (listedabove), of which the first three are the most closely related toICHTHYIN, with up to 57% of homology. Four of these genes have the samebasic structure and between 5 and 8 exons: 1) NIPA1 (Non-Imprinted inPrader-Willi/Angelman syndrome 1) or SPG6 on chromosome 15q11.2; 2)NIPA2 (DUF803.0) also on chromosome 15q11.2; 3) LOC152519 (DUF803.3) onchromosome 4p12; 4) FLJ13955 (DUF803.1) on chromosome 8q22.2; and 5)dJ462023.2 (DUF803.4) on chromosome 1p36.12-p35.1. The FLJ13955 anddJ462023.2 genes are predicted to have 12 exons, share high homology,around 50% with each other, and 25% with the other paralogs. Some ofthese sequences were annotated by automatic analysis pipelines such asGeneWise, Genscan, Gnomon, and could be either too long or incomplete,with the exception of NIPA1 and 2, which have been more completelyanalyzed (19, 20a). Two transcripts were found for NIPA1, one of 2.2 kband a larger one of 7.5 kb with a long 3′UTR which is present at lowlevels in most tissues, but at higher levels in neuronal tissues (19).

Fifty-two homologues were found in eukaryotes including Caenorhabditiselegans, Drosophila, mice and plants (IPR008521;http://ebi.ac.uk/interpro/). Weaker BLASTP homologies were also foundwith a hypothetical transport protein in Pseudomonas denitrificans(ORF6; SW:YCB6_PSEDE) (20b), and with a C. elegans chemoreceptor with 7TM domains of the sri family (WP:CE33629).

Multiple amino acid alignment confirms both the high homology and theconservation of the positions of the TM regions of the six proteins ofthe family (FIG. 3).

The phylogenetic tree (Tree Top,http://www.genebee.msu.su/services/phtree_reduced.html) of the multiplesequence alignments (CLUSTALW) of the six human DUF803 proteins and 347human GPCR protein sequences from the GPCR database (GPCRDB, May 2003release; http://www.gpcr.org/7tm/) identified DUF803 proteins as abranch between glutamate and frizzled/taste2 receptors (21).

The mutations identified in ICHTHYIN include a nonsense mutation in thesecond exon which would lead to the synthesis of a truncated protein.Four of the 5 missense mutations were situated inside one of thepredicted TM domains, in the 2^(nd) (AA80), the 3^(rd) (AA146), the ₄^(th) (AA175),- and the 6^(th) (AA235). The fifth mutation (AA114) islocated between the 2_(nd) and 3^(rd) TM domains.

Genotyping on Chromosomes 15q11.2 and 8q22.2 and Mutation Analysis ofNIPA2 and FLJ13955 in Families in which other Known loci had beenExcluded.

Two of the ICHTHYIN paralogs, NIPA2 and FLJ13955, have been described tobe strongly expressed in keratinocytes (SAGE). In order to test linkage,the localizations containing these paralogs in a form of ARCI, weregenotyped in 65 consanguineous families (226 individuals including 103affected family members): the 52 remaining consanguineous families fromour DNA collection and 13 new consanguineous families. Linkage tochromosome 8q22.2 or 15q 11.2 was suggested in twelve families but whenthe two candidate genes were sequenced for the exons and exon/intronboundaries, no mutations were found.

Method for Screening Agonists and Antagonists

-   1) Cloning of double stranded cDNA: The cloning of ichthyin's    doubled stranded cDNA has been performed using standard methods and    kits (SuperScript Double-Stranded cDNA Synthesis kit). The ichthyin    specific primers for this doubled stranded cDNA are indicated in the    detailed description.-   2) Expression studies in viral vectors: The complete double stranded    cDNA will then be expressed in several vectors after standard    methods for    -   a. The establishment of permanent cell lines,    -   b. The co-transfection experiments with a reporter GPCR gene,    -   c. The analysis of ions (Ca, Cl, H) fluxes,    -   d. The analysis of the expression of the other genes implicated        in ARCI.-   3) Chemical synthesis of (R)-trioxilin A3:    -   a. The metabolites such as (R)-trioxilin A3 will be chemical        synthetized.    -   b. They will be labeled with isotopes in order to performed        binding experiments.    -   c. The biological activity of trioxilin as a key regulator with        induction of experssion of the other genes implicated in ARCI        (Quantitative PCR of RNA from the cells of patients with        mutations in TGM1, ABCA12, ALOX12B, ALOXE3, ABHD5, Ichthyin,        STS, ALDH3A2) will be studied.

4) the screening of agonists and antagonists in the binding experimentswill be performed. TABLE 1 Origin of families and mutations Number Con-of sanguinity Family patients Degree Origin Mutation Effect Exon 1 31^(st) Turkey 703G→A G235R 6 2 1 1^(st) Algeria 247C→T R83X 2 3 1 1^(st)Algeria 247C→T R83X 2 4 1 1^(st) Algeria 247C→T R83X 2 5 2 1^(st)Colombia 341C→A A114N 4 6 1 1^(st) Turkey 341C→A A114N 4 7 3 2^(nd)Turkey 341C→A A114N 4 8 1 1^(st) Turkey 341C→A A114N 4 9 1 1^(st)Algeria 341C→A A114N 4 10 1 1^(st) Algeria 341C→A A114N 4 11 1 1^(st)Algeria 341C→A A114N 4 12 1 1^(st) Algeria 437C→T S146F 5 13 2 1^(st)Syria 523C→G H175N 5 14 4 2^(nd) Algeria 239G→T G80V 2

TABLE 2 Primer sequences for ICHTHYIN and paralogs: exon amplificationand RT-PCR Length Forward Reverse PCR- (bp) of Name sequences sequencesconditions amplicon Exon amplification LOC348938 1 ctcacctcttgcccgccagaacccaga IX 520 ctagc tcttcaa SEQ ID No 10 SEQ ID No 11 2ttatctggcacgtg aggtgggattcca I 595 gtggta gataggg SEQ ID No 12 SEQ ID No13 3 gcctgtgaggaatc ctgggcctcagat I 442 caagag tcacact SEQ ID No 14 SEQID No 15 4 ctccagggagagag ggcctgcctctct I 452 cgtatg attaccc SEQ ID No16 SEQ ID No 17 5 gaacaatgtctccc ccatacatatcag I 599 gtggat gccaggaa SEQID No 18 SEQ ID No 19 6 ttgggggtttaaaa cagttgcactgga II 898 acctaaccaaataacca SEQ ID No 20 SEQ ID No 21 RT-PCR RT_4 gggcaaaggaatataagaggaagtgac VII 1111 cctcatct aaaggcaac SEQ ID No 22 SEQ ID No 23 RT_7ccacgcgggggaca gcaggtgcaaatg VII 474 agtcgc cgtaggctccaaa SEQ ID No 24 gSEQ ID No 25 RT_8 cgtcggcgtgtgcc gcaggtgcaaatg VII 409 ccggcgtaggctccaaa SEQ ID No 26 g SEQ ID No 27 RT_10 ctttggagcctacgggacgaggtaacg VII 574 catttgcac accaccgtgg SEQ ID No 28 SEQ ID No 29Exon amplification paralogs FLJ13955 1 caggctgggagcac gggtgtcttcctg VIII418 ctac agagctg SEQ ID No 30 SEQ ID No 31 2 ccaaacctaccctgatgcactccagcc VI 378 ggaaat tgaatta SEQ ID No 32 SEQ ID No 33 3gcattcccctcata catgacaatggat I 394 gctcag cgctgaa SEQ ID No 34 SEQ ID No35 4 ccacatatcctcag agatggcagctcc I 400 ccacag aagacag SEQ ID No 36 SEQID No 37 5 aatgtggacgctat gggtgcttctgtt I 455 ccaagg gctgact SEQ ID No38 SEQ ID No 39 6 acgttcgagctctg aaccatgagaaag I 379 ggtct gagtagaatgcSEQ ID No 40 SEQ ID No 41 7 gcaacaaggccact ggctgaggacaag I 447 gcttacctacagg SEQ ID No 42 SEQ ID No 43 8 tgactgctgattgg aacctctcctgga VI 389aaatgc agattgtca SEQ ID No 44 SEQ ID No 45 9 agaatgtgtggcataggagctcaggaa I 360 gcaaat actgatgtt SEQ ID No 46 SEQ ID No 47 10ccatgagaaactgg cactgaaaatagc II 371 aagaacaa acctttggtt SEQ ID No 48 SEQID No 49 11 ctgcaccgagcctg tgaaacgccatgt I 300 attatt ctgtagc SEQ ID No50 SEQ ID No 51 12 cttctctttagaga gtgggggaaagga II 374 gtgcccaca ctgaaatSEQ ID No 52 SEQ ID No 53 NIPA2 1 actggggccttgta aggaaaaccgctt V 507aaggaa cagaaca SEQ ID No 54 SEQ ID No 55 1_alternative agtaagctgcctgtcaagaccttggct I 458 cgaagc cagaaaaa SEQ ID No 56 SEQ ID No 57 2tgaagaaatataca cacgaggaatgaa II 491 ggttggtgct gatgtgg SEQ ID No 58 SEQID No 59 3 agaaatcccgagtc agcctgggtgaca IV 346 atgcag gagtgag SEQ ID No60 SEQ ID No 61 4 ctgggcgacagagt aagcgaagttctc III 385 gagact caggttgSEQ ID No 62 SEQ ID No 63 5 gcgaaagccagaat caggcttgggatt II 454 cttcatgatagga SEQ ID No 64 SEQ ID No 65 6_1 gcttgggctgcaaa gacgtctttaaag II599 ataaag gcatgcaa SEQ ID No 66 SEQ ID No 67 6_2 gagcctcatcgtctcgtcatgtgctga II 618 gtgtga ggtcatt SEQ ID No 68 SEQ ID No 69 RT-PCRparalogues FLJ13955 RT3 cttcagctggcaca tggcaccactgtt VII 624 acaagagtcgtat SEQ ID No 70 SEQ ID No 71 RT4 tgattctgctaacc ataacaggccaac VII586 ctggtg agccatc SEQ ID No 72 SEQ ID No 73 NIPA2 RT3 caaggtggccatgcgcaacaagaatat VII 748 atatct ccccacaa SEQ ID No 74 SEQ ID No 75 RT4gagcctcatcgtct cgtcatgtgctga VII 618 gtgtga ggtcatt SEQ ID No 76 SEQ IDNo 77 NIPA1 RT1 ggcccaagacatct gctatccaccaga VII 492 tgcata ccacctg SEQID No 78 SEQ ID No 79 LOC152519 RT1 gggaccctgagtgg ccacctggcagag VII 517attctt acaaag SEQ ID No 80 SEQ ID No 81 DJ462O23.2 RT1 ggaagaagcccattcaggcagaggctg VII 493 ccattt gattcta SEQ ID No 82 SEQ ID No 83PCR conditions:I: As described in Material and Methods;II: TM 55° C.;III: TM 59° C. using Hot Master Taq (Eppendorf);IV: TM 59° C., 100 ng of DNA;V: TM 55° C. using Advantage GC genomic Polymerase Mix (Clontech);VI: TM 55° C., 100 ng of DNA;VII: RT-PCR As described in Material and Methods;VIII: TM 60° C. using Advantage GC genomic Polymerase Mix (Clontech);IX: TM 55° C. using Advantage GC genomic Polymerase Mix (Clontech), 100ng of DNA.

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1-24. (canceled)
 25. A method of treating a skin disorder, comprisingadministering to a subject in need thereof an amount of a compoundselected from a metabolite of 12(R)-hepoxilin pathway or an analogthereof effective for treating said disease.
 26. The method of claim 25,wherein the compound is selected from 12(R)-TXA3, 12(R)-HXA3,12(R)-HXA3-C, 12(R)-HXA3-D and 12(R)-HXA3-E and their ester or amidederivates.
 27. The method of claim 25, wherein the compound is a ligandof an ICHTHYIN protein.
 28. The method of claim 25, wherein the compoundis 12(R)-TXA3 or an agonist or antagonist thereof.
 29. The method ofclaim 25, wherein the skin disorder is selected from a dry skin disorderand an inflammation of the skin.
 30. The method of claim 29, wherein thedry skin disorder is selected from a dermatosis and a keratinizationdisorder.
 31. The method of claim 30, wherein the dermatosis is selectedfrom atopic skin, contact dermatitis, eczema and psoriasis.
 32. Themethod of claim 30, wherein the keratinization disorder is selected froman ichthyosis and a palmoplantar keratoderma.
 33. A compositioncomprising (i) a compound selected from a metabolite of 12(R)-hepoxilinpathway and an analog thereof, and (ii) a pharmaceutically acceptablecarrier for topical application.
 34. The composition of claim 33,wherein the coumpound is 12(R)-TXA3 or an agonist or a synthetic analogthereof.
 35. The composition of claim 33, wherein the coumpound is anagonist or an antagonist of an ICHTHYIN protein.
 36. A method ofdetecting, diagnosing or characterizing the presence of, orpredisposition to a skin disorder in a subject, comprising assessing, ina biological sample from said subject, the presence of a geneticalteration in the ICHTHYIN gene or corresponding protein, the presenceof a genetic alteration in said gene or protein being indicative of thepresence of or predisposition to a skin disorder in said subject. 37.The method of claim 36, wherein the genetic alteration is a mutation, adeletion, an inversion, an addition and/or a substitution of one or moreresidues in said gene or encoded protein.
 38. The method of claim 26,comprising detecting the presence of a mutation in a ICHTHYIN gene orprotein in said biological sample.
 39. The method of claim 36, whereinthe biological sample comprises a tissue, cell or fluid that contains aICHTHYIN gene or polypeptide.
 40. The method of claim 39, wherein thebiological sample comprises genomic DNA from the subject.
 41. The methodof claim 36, wherein the skin disorder is a dry skin disorder.
 42. Themethod of claim 41, wherein the dry skin disorder is selected from adermatosis and a keratinization disorder.
 43. The method of claim 42,wherein the dermatosis is selected from atopic skin, contact dermatitis,eczema and psoriasis.
 44. The method of claim 42, wherein thekeratinization disorder is selected from an ichthyosis and apalmoplantar keratoderma
 45. A method of selecting or identifyingbiologically active compounds useful as candidates for treating a skindisorder, comprising contacting a candidate compound with an ICHTHYIN orrelated protein and determining whether said compound binds to ormodulate the activity of said protein.
 46. The method of claim 45,comprising contacting a test compound with a recombinant host cellexpressing an ICHTHYIN or related protein, and selecting the compoundsthat bind said protein or modulate its activity.